Disorganized Extracellular Matrix Research Articles

Manipulating mitochondrial pyruvate carrier function causes metabolic remodeling in corneal myofibroblasts that ameliorates fibrosis

Myofibroblasts are key cellular effectors of corneal wound healing from trauma, surgery, or infection. However, their persistent deposition of disorganized extracellular matrix can also cause corneal fibrosis and visual impairment. Recent work showed that the PPARγ agonist Troglitazone can mitigate established corneal fibrosis, and parallel in vitro data suggested this occurred through inhibition of the mitochondrial pyruvate carrier (MPC) rather than PPARγ. In addition to oxidative phosphorylation (Ox-Phos), pyruvate and other mitochondrial metabolites provide carbon for the synthesis of biological macromolecules. However, it is currently unclear how these roles selectively impact fibrosis. Here, we performed bioenergetic, metabolomic, and epigenetic analyses of corneal fibroblasts treated with TGF-β1 to stimulate myofibroblast trans-differentiation, with further addition of Troglitazone or the MPC inhibitor UK5099, to identify MPC-dependencies that may facilitate remodeling and loss of the myofibroblast phenotype. Our results show that a shift in energy metabolism is associated with, but not sufficient to drive cellular remodeling. Metabolites whose abundances were sensitive to MPC inhibition suggest that sustained carbon influx into the Krebs’ cycle is prioritized over proline synthesis to fuel collagen deposition. Furthermore, increased abundance of acetyl-CoA and increased histone H3 acetylation suggest that epigenetic mechanisms downstream of metabolic remodeling may reinforce cellular phenotypes. Overall, our results highlight a novel molecular target and metabolic vulnerability that affects myofibroblast persistence in the context of corneal wounding.

Characterization of Immune Cell Infiltration and Collagen Type III Disorganization in Human Secondary Lymphedema: A Case-control Study.

Secondary lymphedema (SL) affects 120 million people globally, posing a lifelong burden for up to 37% of cancer survivors. Chronic inflammation and progressive fibrosis are key drivers of SL, yet detailed characterization of immune cell subpopulations across lymphedema stages is lacking. This study aimed to investigate the immunologic profile of lymphedematous skin and its association with extracellular matrix changes, which could serve as clinical biomarkers or therapeutic targets. This case-control study analyzed the skin from 36 patients with and without SL, using immunofluorescence to quantify T cells, B cells, macrophages, and their subpopulations. Collagen quantity and composition were examined using picrosirius red staining, and mast cell infiltration was assessed with toluidine blue staining. Early and late SL stages were compared to identify histomorphological and immunologic correlates of stage progression. We found a predominance of CD4+ T cells and mast cells in SL skin (1.4/mm² versus 1.0/mm², P < 0.01; 1.2/mm² versus 0.2/mm², P < 0.0001) and a higher ratio of collagen III to collagen I fibers (51.6% versus 75.0%, P < 0.001). M2 macrophages were more abundant in late-stage than in early-stage lymphedema (1.7/mm² versus 1.0/mm², P = 0.02). This study demonstrated a shift toward CD4+ T cell and mast cell infiltration in SL skin, correlating with extracellular matrix disorganization and an altered collagen III/I ratio. These findings enhance our understanding of the cellular and morphological changes in SL, potentially guiding future diagnostic and therapeutic strategies.

Long-Term Follow-Up of Pediatric Excimer Laser-Assisted Penetrating Keratoplasty for Congenital Stromal Corneal Dystrophy.

The purpose of this study was to highlight characteristic clinical and microscopic findings and report the long-term follow-up of pediatric excimer laser-assisted penetrating keratoplasty (excimer-PKP) for congenital stromal corneal dystrophy (CSCD). A 2-year-old Greek child presented with CSCD at our department. Clinical examination showed bilateral flake-like whitish corneal opacities affecting the entire corneal stroma up to the limbus. Genetic testing identified a mutation of the decorin gene (c.962delA). The variant was not present in the parents and represented a de novo mutation. The uncorrected visual acuity was 20/100 in both eyes. Excimer-PKP (8.0/8.1 mm) was performed on the right eye at the age of 2.5 years and on the left eye at the age of 3 years. Postoperatively, alternating occlusion treatment was performed. The light microscopic examination demonstrated a disorganized extracellular matrix of the corneal stroma characterized by a prominent irregular arrangement of stromal collagen lamellae with large interlamellar clefts containing ground substance, highlighted by periodic acid-Schiff- and Alcian blue-positive reaction detecting acid mucopolysaccharides. Electron microscopy showed disorganization and caliber variation of collagen lamellae and thin filaments within an electron-lucent ground substance. The postoperative course was unremarkable. Both grafts remained completely clear 14 years postoperatively. Corneal tomography showed moderate regular astigmatism with normal corneal thickness. The corrected distance visual acuity was 20/25 in both eyes. Excimer-PKP for CSCD might be associated with excellent long-term results and a good prognosis, particularly when the primary surgery is performed at a very young age. However, this requires close postoperative follow-up examinations by an experienced pediatric ophthalmologist to avoid severe amblyopia.

Abstract PO4-25-05: LYVE-1 expressing macrophages modulate the extracellular matrix and contribute to mammary tumor growth

Abstract Macrophages represent a heterogeneous myeloid population with varied functions in normal tissue and tumors. We identify a tissue resident macrophage subpopulation marked by the protein LYVE-1 in both the female mammary gland and mammary tumor. In a genetic LYVE-1+ macrophage deletion model, loss of LYVE-1+ macrophage leads to accumulation of the extracellular matrix glycosaminoglycan hyaluronan. Further, qRT-PCR of tissue lacking LYVE-1+ macrophages yields a gene signature associated with extracellular matrix disorganization. Supporting a tissue remodeling functionality, inducing a LYVE-1 macrophages phenotype in vitro prompts HA internalization and increases hyaluronidase expression. In a murine tumor model, deletion of LYVE-1+ macrophages slows tumor progression and increases HA accumulation, suggesting an anti-tumorigenic, tissue remodeling phenotype. LYVE-1+ macrophages preferentially localize to HA dense regions of tumors, specifically in the tumor stroma, while scRNA-seq of murine tumors demonstrates a LYVE-1+ macrophage phenotype associated with tissue remodeling and immunosuppression. Our data illustrate a LYVE-1+ macrophage population critical for HA homeostasis and tumor progression. Citation Format: Alexis Elfstrum, Annisa Rumahorbo, Braedan McCluskey, Emma Nelson, Kaylee Schwertfeger. LYVE-1 expressing macrophages modulate the extracellular matrix and contribute to mammary tumor growth [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-05.

YAP/TAZ Drive Agrin–Matrix Metalloproteinase 12–Mediated Diabetic Skin Wound Healing

Macroscopic loss of extracellular matrix (ECM) can lead to chronic defects in skin wound healing, but supplementation of ECM holds promise for facilitating wound closure, particularly in diabetic wound healing. We recently showed that the ECM proteoglycan agrin accelerates cutaneous wound healing by improving mechanoperception of migrating keratinocytes and allowing them to respond to mechanical stresses via matrix metalloproteinase-12 (MMP12). RNA-sequencing analysis revealed that in addition to a disorganized ECM, agrin-depleted skin cells have impaired YAP/TAZ transcriptional outcomes, leading us to hypothesize that YAP/TAZ, as central mechanosensors, drive the functionality of agrin-MMP12 signaling during cutaneous wound repair. Herein, we demonstrate that agrin activates YAP/TAZ during migration of keratinocytes post-wounding in vitro and in vivo. Mechanistically, YAP/TAZ sustain agrin and MMP12 protein expression during migration post-wounding through positive feedback. YAP/TAZ silencing abolishes agrin-MMP12 mediated force-recognition and geometrical constraints. Importantly, soluble agrin (sAgrin) therapy accelerates wound closure in diabetic mouse models by engaging MMP12-YAP. Because patients with diabetic foot ulcers and impaired wound healing have reduced expression of agrin-MMP12 that correlates with YAP/TAZ inactivation, we propose that timely activation of YAP/TAZ by sAgrin therapy can accentuate mechanobiological microenvironments for efficient wound healing, under normal and diabetic conditions.

Cronkhite‒Canada syndrome as inflammatory hamartomatous polyposis: new evidence from whole transcriptome sequencing of colonic polyps

BackgroundCronkhite-Canada syndrome (CCS) is a rare, nonhereditary disease characterized by diffuse gastrointestinal polyposis and ectodermal abnormalities. Although it has been proposed to be a chronic inflammatory condition, direct evidence of its pathogenesis is lacking. This study aims to investigate the pathophysiology of CCS by analyzing transcriptomic changes in the colonic microenvironment.MethodsNext-generation sequencing-based genome-wide transcriptional profiling was performed on colonic hamartomatous polyps from four CCS patients and normal colonic mucosa from four healthy volunteers. Analyses of differential expression and multiple enrichment analyses were conducted from the molecular level to the cellular level. Quantitative real-time PCR (qRT-PCR) was carried out to validate the sequencing accuracy in samples from six CCS patients and six healthy volunteers.ResultsA total of 543 differentially expressed genes were identified, including an abundance of CC- and CXC-chemokines. Innate immune response-related pathways and processes, such as leukocyte chemotaxis, cytokine production, IL-17, TNF, IL-1 and NF-kB signaling pathways, were prominently enhanced in CCS colonic polyps. Upregulation of wound healing, epithelial-mesenchymal transition, Wnt, and PI3K-Akt signaling pathways were also observed. Enrichment analyses at different levels identified extracellular structure disorganization, dysfunction of the gut mucosal barrier, and increased angiogenesis. Validation by qRT-PCR confirmed increased expression of the LCN2, IL1B, CXCL1, and CXCL3 genes in CCS colonic polyps.ConclusionsThis case-control whole transcriptome analysis of active CCS colonic hamartomatous polyps revealed intricate molecular pathways, emphasizing the role of the innate immune response, extracellular matrix disorganization, inflammatory cell infiltration, increased angiogenesis, and potential epithelial to mesenchymal transition. These findings supports CCS as a chronic inflammatory condition and sheds light on potential therapeutic targets, paving the way for more effective and personalized management of CCS in the future.

INVESTIGATION OF INTERVERTEBRAL DISC HERNIATION THROUGH A HUMAN ANULUS FIBROSUS PRO-INFLAMMATORY MODEL

Intervertebral disc (IVD) herniation involves a chronic inflammatory process and failure of the annulus fibrosus (AF), in a process still under investigated. Inflammation can lead to disorganization of extracellular matrix (ECM) and fibrotic alterations, among others. Most of the ongoing research relies on in vivo or ex vivo models that do not truly mimic the human microenvironment. Here, we developed a 3D in vitro model aimed at recapitulating inflammation progression in human AF (hAF) to improve the understanding of AF failure.hAF of herniated samples from low back pain patients were collected and digested as previously established. hAF cells were cultured in 2D and 3D, embedded in collagen-type 1 hydrogels. hAF cells in 2D and 3D were then stimulated with IL-1β (10ng/mL), in acute (48h) and chronic (7days) conditions. After 7 days, cell morphology together with extracellular matrix (ECM) (collagen I and fibronectin) were assessed by immunofluorescence. Inflammatory cytokine secretion were analyzed by a protein array.2D experiments showed alteration of cell morphology, with cell area reduction upon inflammatory stimulation. The profile of inflammatory cytokines secreted showed that: i) IL-1α increases with time in culture, while TNF-α decreases; and ii) IL-1α and MIG/CXCL9 are increased in chronic vs acute inflammation. ECM analysis showed no differences. In 3D culture, AF cell morphologic alterations with inflammation were also observed. Concerning ECM production, collagen I production was reduced with chronic inflammation, while fibronectin remained unaltered. Collagen II and aggrecan appear to increase with chronic inflammation. Cytokine production from 3D culture is currently being assessed. ECM alterations are being complemented by characterization at biomechanical (nanoindentation) level.Our data show that chronic inflammation is crucial to understand AF failure mechanisms and improve the resemblance of human IVD herniation.

Extracellular Matrix Disorganization Caused by ADAMTS16 Deficiency Leads to Bicuspid Aortic Valve With Raphe Formation.

A better understanding of the molecular mechanism of aortic valve development and bicuspid aortic valve (BAV) formation would significantly improve and optimize the therapeutic strategy for BAV treatment. Over the past decade, the genes involved in aortic valve development and BAV formation have been increasingly recognized. On the other hand, ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family members have been reported to be able to modulate cardiovascular development and diseases. The present study aimed to further investigate the roles of ADAMTS family members in aortic valve development and BAV formation. Morpholino-based ADAMTS family gene-targeted screening for zebrafish heart outflow tract phenotypes combined with DNA sequencing in a 304 cohort BAV patient registry study was initially carried out to identify potentially related genes. Both ADAMTS gene-specific fluorescence in situ hybridization assay and genetic tracing experiments were performed to evaluate the expression pattern in the aortic valve. Accordingly, related genetic mouse models (both knockout and knockin) were generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method to further study the roles of ADAMTS family genes. The lineage-tracing technique was used again to evaluate how the cellular activity of specific progenitor cells was regulated by ADAMTS genes. Bulk RNA sequencing was used to investigate the signaling pathways involved. Inducible pluripotent stem cells derived from both BAV patients and genetic mouse tissue were used to study the molecular mechanism of ADAMTS. Immunohistochemistry was performed to examine the phenotype of cardiac valve anomalies, especially in the extracellular matrix components. ADAMTS genes targeting and phenotype screening in zebrafish and targeted DNA sequencing on a cohort of patients with BAV identified ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs 16) as a BAV-causing gene and found the ADAMTS16 p. H357Q variant in an inherited BAV family. Both in situ hybridization and genetic tracing studies described a unique spatiotemporal pattern of ADAMTS16 expression during aortic valve development. Adamts16+/- and Adamts16+/H355Q mouse models both exhibited a right coronary cusp-noncoronary cusp fusion-type BAV phenotype, with progressive aortic valve thickening associated with raphe formation (fusion of the commissure). Further, ADAMTS16 deficiency in Tie2 lineage cells recapitulated the BAV phenotype. This was confirmed in lineage-tracing mouse models in which Adamts16 deficiency affected endothelial and second heart field cells, not the neural crest cells. Accordingly, the changes were mainly detected in the noncoronary and right coronary leaflets. Bulk RNA sequencing using inducible pluripotent stem cells-derived endothelial cells and genetic mouse embryonic heart tissue unveiled enhanced FAK (focal adhesion kinase) signaling, which was accompanied by elevated fibronectin levels. Both in vitro inducible pluripotent stem cells-derived endothelial cells culture and ex vivo embryonic outflow tract explant studies validated the altered FAK signaling. Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation.

Abstract 14239: Extracellular Matrix Disorganization Caused by ADAMTS16 Deficiency Leads to Bicuspid Aortic Valve

Background: Extracellular matrix (ECM) is involved in aortic valve development and aortic valve anomaly. ADAMTS gene mediated ECM dysfunction was reported to be related to aortic valve diseases. Hypothesis: The ADAMTS gene family members can contribute to aortic valve diseases. Methods: Morpholino based adamts gene-targeted phenotype-screening of outflow tract anomaly in zebrafish and the 19 ADAMTS family gene-targeted exon sequencing on a cohort BAV patients (304 BAV and 301 normal controls) were performed. Accordingly, both specific genetic tracing and different gene modified mouse models were generated to evaluate the role of specific genes in the aortic valve formation process. Movat’s pentachrome staining was used to describe the altered ECM maturation process. Bulk RNA-seq was performed on both mouse heart tissue and human iPSC-induced ECs to explore the potential molecular pathways, which was further confirmed in ex vivo embryonic outflow tract tissue culture studies. Results: We identified some novel adamts genes that resulted in cardiac outflow valve defects in zebrafish, while exon sequencing identified ADAMTS16 p.H357Q variant in an inherited BAV family. The generated Ad amts16-Cre; Rosa26-tdTomato mice showed Adamts16 -expressing cells were primarily restricted to valvular endothelial cells (VECs) at E12.5 and then expressed in valvular mesenchymal cells (VICs) at E13.5. Both Adamts16 +/- and Adamts16 +/H355Q mice exhibited an R-NC-type BAV phenotype, with progressive aortic valve thickening. Conditional Adamts16 mutant mouse models demonstrated that endothelial lineage cell deficiency in Adamts16 recapitulated the BAV phenotype, which was associated with worse ECM deposition without ECM trilaminar structure. Bulk RNA sequencing unveiled enhanced FAK signaling, which was accompanied by elevated fibronectin levels. Both in vitro iPSC-ECs culture and ex vivo embryonic tissue explant studies validated the altered FAK signaling resulted in exaggerated cell proliferation. Conclusions: Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation.

The Effects of an Osteoarthritic Joint Environment on ACL Damage and Degeneration: A Yucatan Miniature Pig Model.

Posttraumatic osteoarthritis (PTOA) arises secondary to joint injuries and is characteristically driven by inflammatory mediators. PTOA is often studied in the setting of ACL tears. However, a wide range of other injuries also lead to PTOA pathogenesis. The purpose of this study was to characterize the morphological changes in the uninjured ACL in a PTOA inflammatory environment. We retrospectively reviewed 14 ACLs from 13 Yucatan minipigs, 7 of which had undergone our modified intra-articular drilling (mIAD) procedure, which induced PTOA through inflammatory mediators. Seven ACLs were harvested from mIAD minipigs (PTOA) and seven ACLs from control minipigs with no cartilage degeneration (non-PTOA). ACL degeneration was evaluated using histological scoring systems. IL-1β, NF-κB, and TNF-α mRNA expression in the synovium was measured using qRT-PCR. PTOA minipigs demonstrated significant ACL degeneration, marked by a disorganized extracellular matrix, increased vascularity, and changes in cellular shape, density, and alignment. Furthermore, IL-1β, NF-κB, and TNF-α expression was elevated in the synovium of PTOA minipigs. These findings demonstrate the potential for ACL degeneration in a PTOA environment and emphasize the need for anti-inflammatory disease-modifying therapies following joint injury.

Extracellular Matrix Disorganization and Sarcolemmal Alterations in COL6-Related Myopathy Patients with New Variants of COL6 Genes.

Collagen VI is a heterotrimeric protein expressed in several tissues and involved in the maintenance of cell integrity. It localizes at the cell surface, creating a microfilamentous network that links the cytoskeleton to the extracellular matrix. The heterotrimer consists of three chains encoded by COL6A1, COL6A2 and COL6A3 genes. Recessive and dominant molecular defects cause two main disorders, the severe Ullrich congenital muscular dystrophy and the relatively mild and slowly progressive Bethlem myopathy. We analyzed the clinical aspects, pathological features and mutational spectrum of 15 COL6-mutated patients belonging to our cohort of muscular dystrophy probands. Patients presented a heterogeneous phenotype ranging from severe forms to mild adult-onset presentations. Molecular analysis by NGS detected 14 different pathogenic variants, three of them so far unreported. Two changes, localized in the triple-helical domain of COL6A1, were associated with a more severe phenotype. Histological, immunological and ultrastructural techniques were employed for the validation of the genetic variants; they documented the high variability in COL6 distribution and the extracellular matrix disorganization, highlighting the clinical heterogeneity of our cohort. The combined use of these different technologies is pivotal in the diagnosis of COL6 patients.

Loss of RhoE Function in Dermatofibroma Promotes Disorganized Dermal Fibroblast Extracellular Matrix and Increased Integrin Activation

Dermatofibromas are common, benign fibrous skin tumors that can occur at any skin site. In most cases, dermatofibromas are solitary and sporadic, but a few are multiple and familial, and the mechanisms leading to these lesions are currently unclear. Using exome sequencing, we have identified a heterozygous variant in a pedigree with autosomal dominant multiple familial dermatofibromas within RND3 (c.692C>T,p.T231M) that encodes for the small GTPase RhoE, a regulator of the actin cytoskeleton. Expression of T231M-RhoE or RhoE depletion using CRISPR in human dermal fibroblasts increased proliferation, adhesion to extracellular matrix via enhanced β1 integrin activation and more disorganized matrix. The enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was identified as a binding partner for RhoE and formation of this complex was disrupted by T231M-RhoE. PLOD2 promotes collagen crosslinking and activation of β1 integrins, and depleting PLOD2 in T231M-RhoE expressing cells reduced T231M-RhoE-mediated β1 integrin activation and led to increased matrix alignment. Immunohistochemical analysis revealed reduced expression of RhoE but increased expression of PLOD2 in the dermis of dermatofibroma skin samples compared to controls. Our data show loss of RhoE function leads to increased PLOD2 activation, enhancing integrin activation and leading to disorganized extracellular matrix, contributing to dermatofibroma.

Modulation of TRPV4 protects against degeneration induced by sustained loading and promotes matrix synthesis in the intervertebral disc.

While it is well known that mechanical signals can either promote or disrupt intervertebral disc (IVD) homeostasis, the molecular mechanisms for transducing mechanical stimuli are not fully understood. The transient receptor potential vanilloid 4 (TRPV4) ion channel activated in isolated IVD cells initiates extracellular matrix (ECM) gene expression, while TRPV4 ablation reduces cytokine production in response to circumferential stretching. However, the role of TRPV4 on ECM maintenance during tissue-level mechanical loading remains unknown. Using an organ culture model, we modulated TRPV4 function over both short- (hours) and long-term (days) and evaluated the IVDs' response. Activating TRPV4 with the agonist GSK101 resulted in a Ca2+ flux propagating across the cells within the IVD. Nuclear factor (NF)-κB signaling in the IVD peaked at 6 h following TRPV4 activation that subsequently resulted in higher interleukin (IL)-6 production at 7 days. These cellular responses were concomitant with the accumulation of glycosaminoglycans and increased hydration in the nucleus pulposus that culminated in higher stiffness of the IVD. Sustained compressive loading of the IVD resulted in elevated NF-κB activity, IL-6 and vascular endothelial growth factor A (VEGFA) production, and degenerative changes to the ECM. TRPV4 inhibition using GSK205 during loading mitigated the changes in inflammatory cytokines, protected against IVD degeneration, but could not prevent ECM disorganization due to mechanical damage in the annulus fibrosus. These results indicate TRPV4 plays an important role in both short- and long-term adaptations of the IVD to mechanical loading. The modulation of TRPV4 may be a possible therapeutic for preventing load-induced IVD degeneration.

Cytokine profile of human limbal myofibroblasts: Key players in corneal antiviral response.

Corneal transparency may be compromised by viral infections causing corneal scarring, edema, and neovascularization. Ocular injury results from collateral damage induced by exacerbated immune response in corneal stroma. Myofibroblasts play a key role in this process by producing a disorganized extracellular matrix and inflammatory mediators. However, the immune response profile of myofibroblasts during viral infections is still under study. The aim of this work was to analyze the cytokine profile of human limbal myfibroblasts (HLMs) stimulated with the double-stranded RNA analog polyinosinic:polycytidylic acid (poly I:C) and to identify their signaling pathways. HLMs were isolated from cadaveric sclera-corneal rims and stimulated with poly I:C (10μg/ml) for 12h. The secretion of 36 cytokines was measured using the Human Cytokine Array Panel A. The secretion of IFN-β was quantified by ELISA. The expression of pattern recognition receptors (PRRs) such as TLR3, RIG-1 and MDA5 were analyzed by western blot assays. Furthermore, translocation of the nuclear factors NF-κB, IRF3, and IRF7 was assessed by fluorescence staining. In addition, the differentially expressed cytokines were analyzed using the Core Analysis Tool of the Ingenuity Pathway Analysis IPA software. HLMs stimulated with poly I:C increased (fold change&gt;2) the secretion of G-CSF, sTREM-1, CXCL1, CCL1, CXCL8, CXCL10, CXCL11, CCL2, CCL5, IL-13, IL-6, IL-1ra, and IFN-β compared with HLMs under basal conditions. Poly I:C stimulation also induced the expression of RIG-1 (p&lt;0.001), but the expression of TLR3 and MDA5 was unmodified. Finally, HLMs increased nuclear translocation of NF-κB, IRF3, and IRF7 after poly I:C stimulation. Bioinformatic analysis identified canonical signaling pathways associated with cell adhesion and diapedesis, chemokine signaling, and activation of IRFs by cytosolic pattern recognition receptors. These results demonstrate that HLMs secrete cytokines involved in immune cell activation and chemotaxis. The data suggest a key role for HLMs during viral infections in cornea and extend our knowledge about the signaling pathways they trigger.

Elf3 deficiency during zebrafish development alters extracellular matrix organization and disrupts tissue morphogenesis.

E26 transformation specific (ETS) family transcription factors are expressed during embryogenesis and are involved in various cellular processes such as proliferation, migration, differentiation, angiogenesis, apoptosis, and survival of cellular lineages to ensure appropriate development. Dysregulated expression of many of the ETS family members is detected in different cancers. The human ELF3, a member of the ETS family of transcription factors, plays a role in the induction and progression of human cancers is well studied. However, little is known about the role of ELF3 in early development. Here, the zebrafish elf3 was cloned, and its expression was analyzed during zebrafish development. Zebrafish elf3 is maternally deposited. At different developmental stages, elf3 expression was detected in different tissue, mainly neural tissues, endoderm-derived tissues, cartilage, heart, pronephric duct, blood vessels, and notochord. The expression levels were high at the tissue boundaries. Elf3 loss-of-function consequences were examined by using translation blocking antisense morpholino oligonucleotides, and effects were validated using CRISPR/Cas9 knockdown. Elf3-knockdown produced short and bent larvae with notochord, craniofacial cartilage, and fin defects. The extracellular matrix (ECM) in the fin and notochord was disorganized. Neural defects were also observed. Optic nerve fasciculation (bundling) and arborization in the optic tectum were defective in Elf3-morphants, and fragmentation of spinal motor neurons were evident. Dysregulation of genes encoding ECM proteins and matrix metalloprotease (MMP) and disorganization of ECM may play a role in the observed defects in Elf3 morphants. We conclude that zebrafish Elf3 is required for epidermal, mesenchymal, and neural tissue development.

Collagen as a Biomaterial for Skin and Corneal Wound Healing.

The cornea and the skin are two organs that form the outer barrier of the human body. When either is injured (e.g., from surgery, physical trauma, or chemical burns), wound healing is initiated to restore integrity. Many cells are activated during wound healing. In particular, fibroblasts that are stimulated often transition into repair fibroblasts or myofibroblasts that synthesize extracellular matrix (ECM) components into the wound area. Control of wound ECM deposition is critical, as a disorganized ECM can block restoration of function. One of the most abundant structural proteins in the mammalian ECM is collagen. Collagen type I is the main component in connective tissues. It can be readily obtained and purified, and short analogs have also been developed for tissue engineering applications, including modulating the wound healing response. This review discusses the effect of several current collagen implants on the stimulation of corneal and skin wound healing. These range from collagen sponges and hydrogels to films and membranes.

Structure, evolution and expression of zebrafish cartilage oligomeric matrix protein (COMP, TSP5). CRISPR-Cas mutants show a dominant phenotype in myosepta.

COMP (Cartilage Oligomeric Matrix Protein), also named thrombospondin-5, is a member of the thrombospondin family of extracellular matrix proteins. It is of clinical relevance, as in humans mutations in COMP lead to chondrodysplasias. The gene encoding zebrafish Comp is located on chromosome 11 in synteny with its mammalian orthologs. Zebrafish Comp has a domain structure identical to that of tetrapod COMP and shares 74% sequence similarity with murine COMP. Zebrafish comp is expressed from 5 hours post fertilization (hpf) on, while the protein is first detectable in somites of 11 hpf embryos. During development and in adults comp is strongly expressed in myosepta, craniofacial tendon and ligaments, around ribs and vertebra, but not in its name-giving tissue cartilage. As in mammals, zebrafish Comp forms pentamers. It is easily extracted from 5 days post fertilization (dpf) whole zebrafish. The lack of Comp expression in zebrafish cartilage implies that its cartilage function evolved recently in tetrapods. The expression in tendon and myosepta may indicate a more fundamental function, as in evolutionary distant Drosophila muscle-specific adhesion to tendon cells requires thrombospondin. A sequence encoding a calcium binding motif within the first TSP type-3 repeat of zebrafish Comp was targeted by CRISPR-Cas. The heterozygous and homozygous mutant Comp zebrafish displayed a patchy irregular Comp staining in 3 dpf myosepta, indicating a dominant phenotype. Electron microscopy revealed that the endoplasmic reticulum of myosepta fibroblasts is not affected in homozygous fish. The disorganized extracellular matrix may indicate that this mutation rather interferes with extracellular matrix assembly, similar to what is seen in a subgroup of chondrodysplasia patients. The early expression and easy detection of mutant Comp in zebrafish points to the potential of using the zebrafish model for large scale screening of small molecules that can improve secretion or function of disease-associated COMP mutants.

Therapeutic potential of topical administration of acriflavine against hypoxia-inducible factors for corneal fibrosis.

Transdifferentiation of keratocytes into fibroblasts or further into myofibroblasts, which produced denser and more disorganized extracellular matrix, is the major cause of corneal fibrosis and scarring, leading to corneal blindness. TGF-β1 is the critical cytokine for the myofibroblast's transdifferentiation and survival. Hypoxia Inducible Factor (HIF) was found to play an important role in promoting fibrosis in lung, kidney, and dermal tissues recently. Our preliminary study demonstrated that topical administration of the acriflavine (ACF), a drug inhibiting HIF dimerization, delayed corneal opacity and neovascularization after the alkali burn. To know whether ACF could prevent corneal fibrosis and improve corneal transparency, we created a mouse mechanical corneal injury model and found that topical administration of ACF significantly inhibited corneal fibrosis at day 14 post-injury. The reduction of myofibroblast marker α-SMA, and fibronectin, one of the disorganized extracellular matrix molecules, in the corneal stroma were confirmed by the examination of immunohistochemistry and real-time PCR. Furthermore, the ACF inhibited the expression of α-SMA and fibronectin in both TGF-β1 stimulated or unstimulated fibroblasts in vitro. This effect was based on the inhibition of HIF signal pathways since the levels of the HIF-1α downstream genes including Slc2a1, Bnip3 and VEGFA were downregulated. To our knowledge, this is the first time to implicate that HIFs might be a new treatment target for controlling corneal fibrosis in mechanical corneal injuries.

Salzmann’s Nodular Degeneration

Saltzman’s nodular degeneration is a rare, non-inflammatory, slowly progressive degenerative disease of the cornea characterized by bluish-white nodules elevated above the surface of the cornea. More often nodules are located in the mid periphery of the cornea, they can spread into the optical zone. Sometimes localize along the limbus. They consist of dense heterogeneous collagen tissue with hyalinization, localized between the epithelium and Bowman’s layer. Can penetrate deeper into the stroma. The exact pathophysiological mechanism of the lesion is unclear. It is considered that this condition is associated with a violation of the structural relationship of epithelial cells against the background of chronic inflammation of the ocular surface, which leads to the destruction of the Bowman’s layer, the activation and migration of fibroblasts anteriorly, and the deposition of a disorganized extracellular matrix in subepithelial nodules. Clinically, the disease can be manifested by irritation and inflammation of the ocular surface, dryness or decreased vision, more often due to flattening of the cornea and astigmatism. In some cases, the formed tissue can be easily separated from the corneal surface, leaving Bowman’s layer almost intact. In this case, a subsequent phototherapeutic keratectomy may be required to “smooth” the surface. Relapses in these eyes are rare. In other situations (often with extensive peripheral vascularization), the lesions involve the anterior stroma, making it more difficult to remove them while maintaining the smoothness of the cornea. Several laser ablation procedures may be required, but there is a high risk of recurrence with this approach. It may be necessary to treat the meibomian gland’ dysfunction and dry eye syndrome that often accompanies Saltzman’s degeneration to reduce the risk of disease progression.

Extracellular Vesicles in Corneal Fibrosis/Scarring.

Communication between cells and the microenvironment is a complex, yet crucial, element in the development and progression of varied physiological and pathological processes. Accumulating evidence in different disease models highlights roles of extracellular vesicles (EVs), either in modulating cell signaling paracrine mechanism(s) or harnessing their therapeutic moiety. Of interest, the human cornea functions as a refractive and transparent barrier that protects the intraocular elements from the external environment. Corneal trauma at the ocular surface may lead to diminished corneal clarity and detrimental effects on visual acuity. The aberrant activation of corneal stromal cells, which leads to myofibroblast differentiation and a disorganized extracellular matrix is a central biological process that may result in corneal fibrosis/scarring. In recent years, understanding the pathological and therapeutic EV mechanism(s) of action in the context of corneal biology has been a topic of increasing interest. In this review, we describe the clinical relevance of corneal fibrosis/scarring and how corneal stromal cells contribute to wound repair and their generation of the stromal haze. Furthermore, we will delve into EV characterization, their subtypes, and the pathological and therapeutic roles they play in corneal scarring/fibrosis.